ABSTRACT: Currently, the reconstructive procedures used for major tissue loss are inadequate to reconstruct a lost hand or to treat significant injuries to the extremities or face. One potentil solution to the overwhelming need for tissue for reconstruction is the use of Vascularized Composite Allografts (VCA). Currently, nonspecific immunosuppression is the only established method for sustaining these transplants. Immunologic tolerance, however would allow for the long-term survival of these organs without the need for chronic immunosuppression, impacting the risk-benefit ratio, and allowing for more widespread use of VCA in reconstructing lost limbs and facial deformities. In addition, any tolerance strategy developed in a VCA model could be easily applied to other solid organ transplants including those with an epithelized surface such as lung and intestine. One promising method of inducing tolerance is the development of mixed chimerism. Over the past 20 years our laboratory has developed a pre-clinical canine model that offers the integrity of an outbred large animal model with the benefits of defined Dog-Leukocyte Antigen (DLA) system. Studies performed in this model, such as the development of a non-myeloablative hematopoietic stem cell (HSC) transplant regimen, have been clinically translated without the need for testing in primates. While most protocols that use HSC transplantation to induce tolerance towards VCA transplants relies upon on the hypothesis that persistent donor cell chimerism is required for tolerance, previous studies have reported that without HSC and chimerism tolerance cannot be achieved. Our hypothesis, however, challenges this current paradigm in that although chimerism is necessary to induce VCA tolerance, permanent engraftment of donor cells is not necessary for long-term maintenance of tolerance. In other words, the recipient can reject the donor HSC transplant without rejecting the donor VCA. This implies that in a non-life saving VCA transplant such as a hand or a face, elimination of donor chimerism eliminates the risks of graft-vs-host disease (GVHD) and immunodeficiency in the recipient. We believe the immunologic tolerance to vascularized composite allografts can be achieved through a non-myeloablative protocol that emphasizes transient mixed chimerism. The experiments are designed in our large animal pre-clinical model to address the following specific aims. Aim 1: Determine if the infusion of hematopoietic stem cell (HSC) mobilized by Plerixafor (AMD3100), an CXCR4 antagonist, as part of a non-myeloablative protocol combined with a simultaneous transplantation of VCA allograft across a haploidentical mismatch leads to long-term tolerance. Aim 2: Determine if tolerance to mismatched VCAs can be established and maintained with only transient donor cell chimerism. Aim 3: Determine if tolerance to VCA can be established with only transient chimerism through the addition of co-stimulatory blockade to our non- myeloablative protocol. Aim 4: Elucidate the mechanisms in inducing and maintaining VCA tolerance with emphasis on T-regulatory cells, Th1 versus Th2 pathways, and NK cells.